The following references are incorporated herein in their entirety by reference: Certa, H., Fedtke, N., Wiegand. H. J., Miller, A. M. F., Bolt, H. M. Arch. Toxicol. 1996, 71, 112-122; EPA method 604, Phenols in Federal Register, Oct. 26, 1984. Environment Protection Agency, Part VIII, 40 CFR Part 136, 58-66; EPA method 625, Base/neutrals and acids in Federal Register, Oct. 26, 1984. Environment Protection Agency, Part VIII, 40 CFR Part 136, 154-174; Puig, D., Barcelxc3x3. Trends in Anal. Chem, 1996, 15(8), 362-375; Li, N., Lee, H. K. Anal. Chem. 1997, 69, 5193-5199; Bender, M. L., Komiyama, M. Cyclodextrin Chemistry, Springer-Veriag, Berlin, 1978; Breslow, R., Bovy, P., Hersh, C. L. J. Am. Chem. Soc. 1980, 102, 2115; Szejtli, J. Cyclodextrin Technology, Kluwer Academie Publishers, Boston, 1988; Editor Sant""e, D. Minutes of the Sixth International Symposium on Cyclodextrins, Paris, 1992; Editor Bethell, D. Advances in Physical Organic Chemistry, 1994, Volume 29, 1-85, Academic Press. New York; Chen, E. T., Pardue, H. L. Anal. Chem. 1993, 65, 2583-2587; Ikeda, H., Kojin, R., Yoon, C.-L., Ikeda, T., Toda, F., J. Inclusion Phenom. 1989, 7, 117-124; Chen, E. T. unpublished cytotoxicity report of the mM-xcex2-DMCD; Alarie, J. P., Vo-Dinh,T. Talanta, 1991, 38(5), 529-534; Zhao, S., Luong, H. Y. Analytica. Chimica Acta, 1993, 282, 319-327; Liu, H., Li, H., Ying, T., Sun, K., Qin, Y., Qi, D. Analytica. Chimica Acta, 1998, 358, 137-144; Li, G., Mcgown L. B. Dissertation Abstracts International. 1994, 56/02-B; Li, G., Mcgown, L. B. Science, 1994, 264, 249-251; Mallouk, T. E., Harrison, D. J. (editors) Interfacial Design and Chemical Sensing, 1994, ACS Symposium Series 561; Roberts, S. M. Molecular Recognition, Chemical and Biochemical Problems, Royal Society of Chemistry, 1989; Chidsey, C. E. D. Science, 1991, 251, 919-922; Nuzzo, R. G., Fusco, F. A., Allara, D. L. J. Am. Chem. Soc. 1987, 109, 2358-2368; Spasov, A. Ann. Univ. Sufia, II Faculte Phys. Math. Livre, 1939, 2(35), 289-291; Chen, E. T. Dissertation of Ph.D. Title xe2x80x9cA Study of Analytical Application of the Catalytic Properties of Cyclodextronsxe2x80x9d 1994; Chung, C. Dissertation of Ph.D. Title xe2x80x9cSpontaneously Adsorbed Monolayer Films: Fabrication, Characterization, and Application of Monolayers of Alkanethiol and Sulfur-bearingxe2x80x9d, 1990; Markowitz, M. A., Bielski, R., Regen, S. L. J. Am. Chem, Soc.,1988, 110, 7545-7546; Gregg, B. A., Heller, A. J. Phys. Chem. 1991, 95, 5976-5980; Komiyama, M. Ange. Macromole. Chemie, 1988, 163, 205-207; Koradecki, D., Kutner, W. J. Incl. Phenom. 1991, 10, 79-96; Lehn, J. M. Science, 1985, 227, 849; Proceedings of the NATO Advanced Research Workshop on Chemosensors of Ion and Molecular Recognition, Kluwer Academic Publishers, Bonas, France, 1997; (Editors) Scheller, F. W., Schubert, F., Fedrowitz, J. Frontiers in Biosensorics, (books one and two), Birkh-user Verlag Base, Boston, 1997; Szejtli, J., Szente, L. Proceedings of the Eighth International Symposium on Cyclodextrins, Budapest, Hungary, Kluwer Academic Publishers, Boston, 1996; Dagani, R. C and EN. Jun. 8, 1998, 35-46; Pardue, H. L. Anal. Chim. Acta, 1989, 216, 69-107; Williams, M., Pardue, H. L, Uhefbu, C. E., Smith, A. M., Studley, J. Talanta, 1996, 43, 1379-1385; Lim, K. B., Pardue, H. L. Anal. Chim. Acta, 1996, 329, 285-295; Wang, X., Pardue, H. L. Anal. Chem. 1997, 69, 4482-4489; Kotte, H., Grundig, B., Vortop, K-D., Strehlitz, B., Stottmeister,U. Anal. Chem. 1995, 67, 65-70; Bucke, C. Polysaccharide biotechnology-a Cinderella subject, Trends in Biotech. 1998, 16(2), 50-52; Ross, et al. Arch. Pathol. Lab. Med. 1998, 122:587-608; Wang, J. Anal. Chim. Acta 1997, 337:41; and Biosensors and Electronic Noses, Kres-Roger, Editor, CRC Press, N.Y., 1997.
The present invention relates to the field of biosensors and, in particular, to biosensors comprising a catalytically active cyclodextrins.
Many chemicals in common use in industrialized societies contain aromatic esters. Examples of the types of chemicals containing aromatic esters include detergents, antioxidants and agricultural chemicals. Upon degradation of these aromatic esters whether through enzymatic hydrolysis or bacterial degradation, toxic phenols and phenol derivatives are produced. Research has shown that these toxic chemicals can accumulate in food, soil, and water. In addition, it has been shown that the presence of these chemicals can be dangerous to humans and animals as they can have adverse effects on reproduction and have been implicated in the development of tumors (Certa et al. 1996). The United States Environmental Protection Agency (US-EPA) has listed phenolic compounds as priority pollutants due to their toxicity and persistence in the environment (EPA method 604, Phenols in Federal Register, Oct. 26, 1984, Environment Protection Agency, Part VIII, 40 CFR Part 136, 58-66; EPA method 625, Base/neutrals and acids in Federal Register, Oct. 26, 1984. Environment Protection Agency, Part VIII, 40 CFR Part 136, 154-174). Furthermore, European Community Directive 76/464/EEC recommends that the maximum level of phenolic compounds in surface water for drinking purposes should be in the 1-10 xcexcg/L range (Puig et al.). Therefore, developing a sensitive, reliable, and fast testing method for the detection of phenolic compounds is an issue of importance to the entire industrialized world.
Current methods for the detection of phenolic compounds include liquid chromatography with electrochemical (LCEC) detection and a coupled gas chromatography/mass spectrometry (GC/MS) method which requires sample pretreatment (Puig et al. 1996; Li et al. 1997). These currently employed methods suffer from various limitations. For example, the LCEC method is subject to interference because of the high applied potential (around 1V) required for electrochemical detection of the phenolic compounds. The high polarizing potential causes oxidation of other matrix compounds; hence, an increase in background current is frequently observed. In addition, the LCEC method has problems with signal stability, pH dependence, and time consuming experimental protocols. The GC/MS method usually requires sample derivatization prior to analysis. For example, Li and co-workers (Li et al. 1997) converted phenols to phenyl acetate prior to analyzing with GC/MS. It has been suggested by Puig (Puig et al. 1996) that the US-EPA method for derivatization of nitrophenols for GC/MS may often lead to incorrect results.
Conventional electrochemical methods used to detect toxic phenols suffer from signal drift, and the probes need frequent cleaning because of polymerization caused by oxidation of phenols (Puig et al. 1996). Because traditional electrochemical methods are sensitive to pH they have limited practical application. To date, no satisfactory approach exists for measuring phenols. Cyclodextrins (CDs) and modified CDs have been used as biomimetic enzyme (BMZ) catalysts for several decades (Bender et al. 1978; Breslow et al. 1980; Szejtli et al. 1988; Editor Sant""e, D. Minutes of the Sixth International Symposium on Cyclodextrins, Paris, 1992; Editor Bethell, D. Advances in Physical Organic Chemistry, 1994, Volume 29, 1-85, Academic Press, N.Y.). CDs are cyclic carbohydrates made up of six (xcex1-CD), seven (xcex2-CD) or eight (xcex3-CD) linked D-glucopyranose units. They look like hollow truncated cones, where the interior cavity is hydrophobic and the outside is hydrophilic. The cavities can entrap a variety of chemicals having suitable size and hydrophobicity. Functional groups can be attached to the CDs enabling them to mimic enzyme catalysis. For example, one or two imidazolyl groups attached on the C-3 position of the dimethyl-xcex2-cyclodextrin (xcex2-DMCD) can enhance catalysis of the hydrolysis of paranitrophenyl acetate (p-NPA) to para-nitrophenolate (p-NPOxe2x88x92) with rate increases up to several thousand times the un-catalyzed rate (Chen et al. 1993; Ikeda et al. 1989). The nomenclature used to identify the imidazole modified xcex2-DMCDs is mM-xcex2-DMCD for mono-imidazolyl substituted xcex2-DMCD and bM-xcex2-DMCD for bis-imidazolyl substituted xcex2-DMCD. The M before xcex2 in the abbreviation represents imidazolyl group. mM-xcex2-DMCD has been used in solution to mimic the natural enzyme xcex2-chymotrypsin. The protease xcex2-chymotrypsin has a pH optimum of 8.2 for the hydrolysis reaction of p-NPA and achieves only a modest rate acceleration at this pH. In contrast, mM-xcex2-DMCD can work at wide range of pH values. In addition, these modified CDs have good stability, and have unique solubility in both aqueous and organic phases. The mM-xcex2-DMCD showed good selectivity for p-NPA and the cytotoxicity of mM-xcex2-DMCD has been studied (Chen et al. 1993; Ikeda et al. 1989; Chen, E. T. unpublished cytotoxicity report of the mM- -DMCD).
Biosensors of the prior art generally contain immobilized enzymes on the surface of an electrode. This type of biosensor has found application for the detection of various analytes. Systems of this type generally include a mediator that functions to shuttle electrons from the electrode to the electrochemically active species detected. The biosensors of the prior art based on immobilized enzymes have a major flaws in that the response time is dependent upon the concentration of the analyte and the requirement for a mediator introduces an additional complexity and source of error.
New types of biosensors have been developed utilizing CDs and CD derivatives. The unique properties of CDs have been used to enhance the performance of biosensors with both optical and electrochemical detection. Examples of the use of CDs in sensors are provided by U.S. Pat. No. 5,540,828 to Yacynych, U.S. Pat. Nos. 5,587,466 and 5,480,924 issued to Vieil, et al. and U.S. Pat. No. 5,432,274 issued to Luong, et al., the specifications of which are specifically incorporated herein by reference. A variety of analytes can be detected in a fast, selective and sensitive way using CDs. Alarie and co-workers have developed a fiber-optic CD-based fluorescence sensor that utilized CDs"" inclusion property to detect pyrene (Alarie et al. 1991). When using traditional electrochemical methods, electron mediators are needed in most cases; however, most of the mediators are toxins. Luong and co-workers used modified CDs to form a water soluble complex with tetrathiafulvalene (TTF) and used the complex as an electron mediator for a glucose biosensor (Zhao et al. 1993). The inclusion property of CDs was used in the development of an amperometric glucose biosensor as reported by Liu and coworkers (Liu et al. 1998). Recently, CDs, together with inclusion compounds, were found to form molecular nanotubes through self-assembly (Li et al., Dissertation Abstracts International 1998; Li et al., Science 1994). Molecular self-assembly technology for developing membranes is recognized as superior to conventional techniques because it can provide varying degrees of spatial and orientation arrangements of amphiphilic molecules on variety of surfaces of substrates as reported elsewhere (Mallouk et al. 1994; Roberts 1989; Chidsey 1991; Nuzzo et al. 1987). The formation of nanotubes made with CD and diphenylhexatriene based on the molecular inclusion has reported in the literature (Le et al. 1994).
One analytical technique which may be used in conjunction with a biosensor is cyclic voltammetry. In cyclic voltammetry, the potential of the electrode is scanned linearly from an initial value to a second value and then back to the initial value or some other potential. As the potential is scanned in the positive direction, an anodic current occurs when the electrode becomes a sufficiently strong oxidant to oxidize the analyte. The anodic current increases rapidly until the concentration of the analyte on the electrode surface approaches zero corresponding to a peak in the current. The current then decays as the solution surrounding the electrode is depleted of the analyte due to the conversion of the analyte into an oxidized form. When the highest potential of the scan is reached, the potential is scanned in the negative direction. When the electrode becomes a sufficiently strong reductant, the oxidized form of the analyte is reduced back to the original form. This reduction causes a cathodic current that increases until the concentration of the oxidized form of the analyte on the electrode approaches zero at which point the current peaks. The cathodic current then decays as the solution of the in the vicinity of the electrode is depleted of the oxidized form of the analyte. The cycle is completed when the potential returns to the initial value or to another predetermined potential value. Additional scans may then be made. When the oxidized form of the analyte is not reduced during the scan back to the starting potential, the reaction is said to be irreversible. The parameters determined in a cyclic voltammogram are the magnitude of the anodic peak current, ipa, the anodic peak potential, Epa, the cathodic peak current, ipc, and the cathodic peak potential, Epc. The pseudo first order rate constants can be obtained from plots of the ln(i∞-it) where i∞ is the maximum current and it is the current at time t.
Notwithstanding the above mentioned uses of CDs, utilizing the catalytic and molecular recognition features of mM-xcex2-DMCD for biosensor development is difficult for the following reasons: (1) low coverage due to the monolayer defects, (2) low sensitivity and (3) low reproducibility as reported in the literature (Chung 1990; Gregg et al. 1991; Komiyama 1988; Koradecki et al. 1991). Thus, there exists a need in the art for a biosensor specific for phenolic compounds. In addition, there exists a need in the art for biosensors that do not utilize electron mediator molecules. These and other needs have been met by the present invention.
It is an object of the present invention to provide a novel biosensor. In preferred embodiments, the biosensor of the present invention may comprise an electrode and a catalytically active cyclodextrin affixed thereto.
It is an object of the present invention to provide a biosensor specific for the detection of phenolic compounds. In preferred embodiments, the biosensor of the present invention may comprise a xcex2-DMCD comprising one or more imidazole groups. In a most preferred embodiment, the biosensor of the present invention may comprise a mM-xcex2-DMCD.
It is an object of the present invention to provide a biosensor capable of detecting molecules of interest that does not require the inclusion of a mediator.
It is an object of the present invention to provide a method for detecting an analyte of interest comprising the steps of contacting a solution containing the analyte with a biosensor and detecting the analyte wherein the biosensor comprises a catalytically active cyclodextrin.
It is an object of the present invention to provide a method of detecting the presence of o-NPA in solution comprising the step of contacting a solution containing o-NPA with a biosensor, which biosensor comprises a catalytically active cyclodextrin. In preferred embodiments, the cyclodextrin may be mM-xcex2-DMCD.
The present invention provides a novel biosensor comprising an electrode with a catalytically active cyclodextrin attached thereto. In a preferred embodiment, the novel biosensor of the present invention comprises a modified cyclodextrin capable of catalyzing the hydrolysis of NPA thereby making possible the measurement of nitrophenyl acetate (NPA) without the use of an electron mediator. In other preferred embodiments, the catalytically active cyclodextrins of the present invention may be assembled in the form of nanotubes.
The electrode of the present invention may be constructed of any material customarily used by those skilled in the art for the construction of electrodes. In preferred embodiments, the electrode may be glassy carbon, gold or silver. In a most preferred embodiment, the electrode may be glassy carbon.
The biosensor of the present invention may be constructed by coating the surface of an electrode with a catalytically active cyclodextrin to form a membrane. This coating may be accomplished by any means known by those skilled in the art. In addition to a cyclodextrin, the electrode may be coated with one or more compounds. In preferred embodiments, the electrode may be coated with a catalytically active cyclodextrin and a polyethylene glycol (PEG). In another preferred embodiment, the electrode may be coated with a catalytically active cyclodextrin, a PEG and a polyvinylpyridine (PVP). In a most preferred embodiment, the cyclodextrin will be deposited in the form of nanotubes and will be applied by co-polymerization of mM-xcex2-DMCD with polyethylene glycol diglycidyl ether (PEG) and poly(4-vinylpyridine) (PVP).